Equine infectious anemia virus (EIAV) is a lentivirus distantly related to the human and simian immunodeficiency viruses (HIV and SIV). Our aim is to identify and characterize the cis- and trans-acting components of pathways that regulate EIAV gene expression. These studies will also broaden our understanding of HIV gene regulation. cDNA libraries were constructed from different cell lines persistently infected with EIAV. Several positive clones representing regulatory gene mRNAs were isolated and subcloned for nucleotide sequencing and to examine function in transfected cells. Nucleotide sequence analysis showed that the predominant mRNAs in each cell line were different. In one cell line, the mRNA had 3 exons while the other expressed a 4 exon message. The first 2 exons were the same in all clones; they were derived from the 5' end and central region of the virus and encoded the Tat protein. The 3 exon clones lacked an exon from the 5' end of the env gene that is spliced to an open reading frame (ORF) from the 3' end of the virus. This ORF is likely to encode a Rev protein; its product was detected in infected cells with antipeptide antisera. The deduced amino acid sequence of the Tat ORF revealed a protein with two domains very similar to regions of HIV and SIV Tat proteins; however, the EIAV protein lacks a cysteine-rich domain shared by the primate virus proteins. Mutagenesis of the tat gene revealed that translation is initiated at a non-AUG codon; a CUG codon from exon I appears to be used instead. Chimeric EIAV/HIV Tat proteins have been constructed; we are taking advantage of differences in promoter specificity to define the functions of the protein domains.